17‐β‐estradiol reduces surface PD‐L1 expression in estrogen receptor‐positive breast cancer but not type 1 endometrial cancer cells

We report here that 17- β -estradiol (E2), through ER α (estro-gen receptor α ), inhibits IFN- γ -induced surface PD-L1 (programmed death-ligand 1, CD274) level in advanced ER + /HER2 − breast cancer (BC) but not in triple negative BC (TNBC) or endometrial cancer (EC). This study implies that mTOR and MAPK pathways can reduce the surface PD-L1 level and diminish immune evasion via ER α . It also has important implications on endocrine resistance and the limitations of mTOR or MAPK-targeted therapies for treating advanced ER + /HER2 – BC.

Since IFN-γ is a major driver of surface PD-L1 expression through the IFN-γ-JAK-STAT-IRF1 pathway in multiple cancers, 7 we exposed these cells to different concentrations of IFN-γ for 24 h and demonstrated that IFN-γ strongly increased the surface PD-L1 level in MCF-7, MDA-MB-231 and TEN. This did not happen to Ishikawa ( Figure 1A-D), even after 3 days of exposure to 50 ng/ml IFN-γ ( Figure 1G,H). Comparatively, MCF-7 showed a strong response, with the surface PD-L1 level peaking at 48 h and declining non-significantly at 72 h ( Figure 1E,F). The same trend occurred after the cells were exposed to 10 ng/ml IFN-γ in a phenol-red free medium containing 5% CSFBS (charcoal-stripped fetal bovine serum) for 3 days ( Figure 1I,J).
We exposed the four cell lines to 10 ng/ml IFN-γ, 10 nM E2, and a combination of both in a phenol-red free medium containing 5% CSFBS during an eight-day culture. We showed that E2 alone did not alter the PD-L1 level on the surface of all four cell lines but greatly downregulated the IFN-γ-induced PD-L1 at the surface and transcriptional levels in MCF-7 co-exposed to E2 and IFN-γ, compared with IFN-γ exposure alone (Figure 2A-F), suggesting that it is not endogenous but the IFN-γ-induced surface PD-L1 that was downregulated by E2 in the advanced ER + /HER2 − BC in vitro.
As E2 affected ERα + /ERβ + MCF-7 instead of the ERα − /ERβ + MDA-MB-231, we used two ERα antagonists tamoxifen ( Figure 3A) and ICI 182,780 (Fulvestrant) ( Figure 3B), and the ERβ antagonist PHTPP ( Figure 3C) to block both receptors, respectively, in MCF-7. We demonstrated that ERα contributed to the E2's downregulation at the surface and transcriptional ( Figure 3D) levels. Since the PD-L1 level in the ERα − /ERβ + A549 lung cancer cell line is strongly upregulated by IFN-γ via the JAK/STAT/IRF-1 pathway, 8 our outcome was further validated in this line that ERβ was not involved ( Figure 3E). F I G U R E 1 IFN-γ's effect on surface PD-L1 expression in MCF-7 (A, E, F, I and J), MDA-MB-231 (B), Ishikawa (C, G and H) and TEN (D) for indicated times. The surface PD-L1 levels were assessed by flow cytometry in both BC and EC cell lines exposed or not exposed to different concentrations of human IFN-γ recombinant protein at indicated time points. Non-filled (open) peaks and grey peaks: The cancer cells exposed or not exposed to IFN-γ were stained with the APC-conjugated mouse IgG1K isotype control antibodies. Orange and pink peaks: The non-IFN-γ exposed cancer cells were labeled with APC-conjugated anti-human CD274 antibodies. Filled blue, purple and green peaks: The cells exposed to IFN-γ were labeled with the APC-conjugated anti-human CD274 antibodies. show the surface PD-L1 levels in both Ishikawa and MCF-7 exposed or not exposed to IFN-γ for 3 days. The lower panel of (I): Representative dot plots display the frequencies of the surface PD-L1 expressing cells (PD-L1 + cells). The numbers denote the percentages of the gated surface PD-L1 + population in total live cells. Data are presented as mean ± SEM. Two-way ANOVA was employed with multiple comparisons of Tukey for the analyses of statistics. *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001. ns: not significant. F I G U R E 2 E2's effect on IFN-γ-induced surface PD-L1 expressions in the cell lines of MCF-7, MDA-MB-231, Ishikawa and TEN. Three days after seeding, 10 nM E2 was added to the four cell lines maintained in a phenol-red-free medium containing 5% CSFBS. 10 ng/ml IFN-γ either alone or combined with 10 nM E2 was applied to the cells on day six. 48 hours later (on the eighth day), the above cells exposed to E2, IFN-γ, or E2 plus IFN-γ and the vehicle controls were harvested for flow cytometric analysis or qRT-PCR to determine surface PD-L1 or PD-L1 mRNA levels. Pure ethanol and solvent of IFN-γ (PBS, pH 7.2 plus 0.5% BSA) were used as the negative (vehicle) controls. (A-D) Upper panel: Representative overlay histograms of the surface PD-L1 expression in each cell line exposed or not exposed to E2, IFN-γ or the combination of both. Our finding accords with a newly published work by Hühn et al., 9 who showed that total E2 deprivation or fulvestrant treatment increased the surface PD-L1 expression in MCF-7 through ERα. Their report designated changes in MFI, however, we observed changes in the proportion (%) of the PD-L1 expressing cells. This suggests that E2 may protect ER + /HER2 -BC by interacting with ERα and reducing the quantity of IFN-γ-induced surface PD-L1 expressing cells. Thus, such a protective role by E2 may slow the progression of ER + BC's deterioration. This may explain why ER + /HER2 -BC expresses less PD-L1, less malignance than the basal TNBC subtype, and less sensitivity to anti-PD-1/PD-L1 therapy than ER − BC.
We confirmed that the JAK/STAT/IRF1 pathway regulates the IFN-γ-induced surface PD-L1 level (Figure 4,1  and 2A). Co-exposure to BEZ235 or U0126 combined with E2 and IFN-γ significantly restored the proportion of the E2-downregulated IFN-γ-induced surface PD-L1 F I G U R E 3 Role of ERα in E2-downregulated-IFN-γ-induced surface PD-L1 level in MCF-7. On the second day after seeding the MCF-7 cells in a phenol-red-free medium containing 5% CSFBS, 10 nM E2 and different concentrations of ER antagonists were added, either alone or in combination. On the third day, 10 ng/ml IFN-γ was added. 48 hours later, the cells exposed to E2, IFN-γ, ERα antagonists of tamoxifen The A549 cells were exposed to E2 and IFN-γ either alone or in combination for 48 h under the five-day culture condition, proving that ERβ does not influence E2's reducing effect (n = 3). Data are presented as mean ± SEM. One-way ANOVA was employed with multiple comparisons of Tukey for statistics. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 and ****p ≤ 0.0001. ns: not significant. expressing cells in MCF-7 ( Figure 4,2C), but LY294002 or other kinase inhibitors (Table S5) were not capable (Figure 4,2B,C). Additionally, BEZ235 performed at a much greater recovery rate than U0126, significantly higher than those exposed to IFN-γ in MCF-7 (Figure 4,2D). This outcome implies that both the mTOR and the MAPK pathways cooperate in inhibiting immune evasion through ERα by reducing the IFN-γ-induced surface PD-L1 expression in ER + BC.
We found that tamoxifen or ICI 182,780 potentiates IFN-γ to upregulate surface PD-L1 expression, even when E2 is present in MCF-7 ( Figure 3A,B). The higher the antiestrogen dose, the greater the PD-L1 surface expression ( Figure 3A,B) as ICI 182, 780 inhibited ER in a dose-dependent manner. 10 This indicates that prolonged administration of antiestrogens may induce tumour cell surface PD-L1 expression by disrupting a dynamic protec-tive effect built by the E2-ERα-IFN-γ-mTOR-MAPK axis in the tumour milieu of the advanced ER + /HER2 -BC patients, triggering tumour immune evasion. Together, these findings may provide causal reasons for antiestrogen resistance and the limited efficacy of mTOR or MAPKtargeted therapies for treating advanced ER + /HER2 -BC patients. Additionally, the increased tumour cell surface PD-L1 induced by the inhibitors of ERα, mTOR or MAPK may potentially augment the sensitivity of ER + BC cells to immune checkpoint inhibitors.

CONCLUSION
E2 reducing the IFN-γ-induced cancer cell surface PD-L1 may be the main reason why high-level PD-L1 protein in ER + BC tumours is rare. Antiestrogens or targeted The mTOR pathway has stronger restorative effects than the MAPK pathway. Data are presented as mean ± SEM. One-way ANOVA was employed with multiple comparisons of Tukey for statistical analyses. *p ≤0.05; ***p ≤ 0.001 and ****p ≤ 0.0001. ns: not significant. VC: vehicle control.
therapies combined with anti-PD-1/PD-L1 regiments could be more beneficial in treating advanced ER + /HER2 -BC patients.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.